Changes in Parasite Virulence Induced by the Disruption of a Single Member of the 235 kDa Rhoptry Protein Multigene Family of Plasmodium yoelii

Invasion of the erythrocyte by the merozoites of the malaria parasite is a complex process involving a range of receptor-ligand interactions. Two protein families termed Erythrocyte Binding Like (EBL) proteins and Reticulocyte Binding Protein Homologues (RH) play an important role in host cell recognition by the merozoite. In the rodent malaria parasite, Plasmodium yoelii, the 235 kDa rhoptry proteins (Py235) are coded for by a multigene family and are members of the RH. In P. yoelii Py235 as well as a single member of EBL have been shown to be key mediators of virulence enabling the parasite to invade a wider range of host erythrocytes. One member of Py235, PY01365 is most abundantly transcribed in parasite populations and the protein specifically binds to erythrocytes and is recognized by the protective monoclonal antibody 25.77, suggesting a key role of this particular member in virulence. Recent studies have indicated that overall levels of Py235 expression are essential for parasite virulence. Here we show that disruption of PY01365 in the virulent YM line directly impacts parasite virulence. Furthermore the disruption of PY01365 leads to a reduction in the number of schizonts that express members of Py235 that react specifically with the mcAb 25.77. Erythrocyte binding assays show reduced binding of Py235 to red blood cells in the PY01365 knockout parasite as compared to YM. While our results identify PY01365 as a mediator of parasite virulence, they also confirm that other members of Py235 are able to substitute for PY01365

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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A Role for Immune Responses against Non-CS Components in the Cross-Species Protection Induced by Immunization with Irradiated Malaria Sporozoites

Immunization with irradiated Plasmodium sporozoites induces sterile immunity in rodents, monkeys and humans. The major surface component of the sporozoite the circumsporozoite protein (CS) long considered as the antigen predominantly responsible for this immunity, thus remains the leading candidate antigen for vaccines targeting the parasite's pre-erythrocytic (PE) stages. However, this role for CS was questioned when we recently showed that immunization with irradiated sporozoites (IrrSpz) of a P. berghei line whose endogenous CS was replaced by that of P. falciparum still conferred sterile protection against challenge with wild type P. berghei sporozoites. In order to investigate the involvement of CS in the cross-species protection recently observed between the two rodent parasites P. berghei and P. yoelii, we adopted our gene replacement approach for the P. yoelii CS and exploited the ability to conduct reciprocal challenges. Overall, we found that immunization led to sterile immunity irrespective of the origin of the CS in the immunizing or challenge sporozoites. However, for some combinations, immune responses to CS contributed to the acquisition of protective immunity and were dependent on the immunizing IrrSpz dose. Nonetheless, when data from all the cross-species immunization/challenges were considered, the immune responses directed against non-CS parasite antigens shared by the two parasite species played a major role in the sterile protection induced by immunization with IrrSpz. This opens the perspective to develop a single vaccine formulation that could protect against multiple parasite species

Gene-Specific Signatures of Elevated Non-Synonymous Substitution Rates Correlate Poorly across the Plasmodium Genus

BACKGROUND: Comparative genome analyses of parasites allow large scale investigation of selective pressures shaping their evolution. An acute limitation to such analysis of Plasmodium falciparum is that there is only very partial low-coverage genome sequence of the most closely related species, the chimpanzee parasite P. reichenowi. However, if orthologous genes have been under similar selective pressures throughout the Plasmodium genus then positive selection on the P. falciparum lineage might be predicted to some extent by analysis of other lineages. PRINCIPAL FINDINGS: Here, three independent pairs of closely related species in different sub-generic clades (P. falciparum and P. reichenowi; P. vivax and P. knowlesi; P. yoelii and P. berghei) were compared for a set of 43 candidate ligand genes considered likely to be under positive directional selection and a set of 102 control genes for which there was no selective hypothesis. The ratios of non-synonymous to synonymous substitutions (dN/dS) were significantly elevated in the candidate ligand genes compared to control genes in each of the three clades. However, the rank order correlation of dN/dS ratios for individual candidate genes was very low, less than the correlation for the control genes. SIGNIFICANCE: The inability to predict positive selection on a gene in one lineage by identifying elevated dN/dS ratios in the orthologue within another lineage needs to be noted, as it reflects that adaptive mutations are generally rare events that lead to fixation in individual lineages. Thus it is essential to complete the genome sequences of particular species of phylogenetic importance, such as P. reichenowi

Plasmodium falciparum Merozoite Invasion Is Inhibited by Antibodies that Target the PfRh2a and b Binding Domains

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes

Physiological modeling, tight glycemic control, and the ICU clinician: what are models and how can they affect practice?

Critically ill patients are highly variable in their response to care and treatment. This variability and the search for improved outcomes have led to a significant increase in the use of protocolized care to reduce variability in care. However, protocolized care does not address the variability of outcome due to inter- and intra-patient variability, both in physiological state, and the response to disease and treatment. This lack of patient-specificity defines the opportunity for patient-specific approaches to diagnosis, care, and patient management, which are complementary to, and fit within, protocolized approaches

Bovine cryptosporidiosis: impact, host-parasite interaction and control strategies

International audienceAbstractGastrointestinal disease caused by the apicomplexan parasite Cryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. At present, effective therapeutic and preventative measures are not available and a better understanding of the host–pathogen interactions is required. Cryptosporidium parvum is also an important zoonotic pathogen causing severe disease in people, with young children being particularly vulnerable. Our knowledge of the immune responses induced by Cryptosporidium parasites in clinically relevant hosts is very limited. This review discusses the impact of bovine cryptosporidiosis and describes how a thorough understanding of the host–pathogen interactions may help to identify novel prevention and control strategies

Characterization of the repertoire diversity of the Plasmodium falciparum stevor multigene family in laboratory and field isolates.

BACKGROUND: The evasion of host immune response by the human malaria parasite Plasmodium falciparum has been linked to expression of a range of variable antigens on the infected erythrocyte surface. Several genes are potentially involved in this process with the var, rif and stevor multigene families being the most likely candidates and coding for rapidly evolving proteins. The high sequence diversity of proteins encoded by these gene families may have evolved as an immune evasion strategy that enables the parasite to establish long lasting chronic infections. Previous findings have shown that the hypervariable region (HVR) of STEVOR has significant sequence diversity both within as well as across different P. falciparum lines. However, these studies did not address whether or not there are ancestral stevor that can be found in different parasites. METHODS: DNA and RNA sequences analysis as well as phylogenetic approaches were used to analyse the stevor sequence repertoire and diversity in laboratory lines and Kilifi (Kenya) fresh isolates. RESULTS: Conserved stevor genes were identified in different P. falciparum isolates from different global locations. Consistent with previous studies, the HVR of the stevor gene family was found to be highly divergent both within and between isolates. Importantly phylogenetic analysis shows some clustering of stevor sequences both within a single parasite clone as well as across different parasite isolates. CONCLUSION: This indicates that the ancestral P. falciparum parasite genome already contained multiple stevor genes that have subsequently diversified further within the different P. falciparum populations. It also confirms that STEVOR is under strong selection pressure

<i>Plasmodium falciparum</i> Replication factor C subunit 1 is involved in genotoxic stress response

AbstractAbout half the world’s population is at risk of malaria, with Plasmodium falciparum malaria being responsible for the most malaria related deaths globally. Antimalarial drugs such as chloroquine and artemisinin are directed towards the proliferating intra-erythrocytic stages of the parasite, which is responsible for all the clinical symptoms of the disease. These antimalarial drugs have been reported to function via multiple pathways, one of which induces DNA damage via the generation of free radicals and reactive oxygen species. An urgent need to understand the mechanistic details of drug response and resistance is highlighted by the decreasing clinical efficacy of the front line drug, Artemisinin.The replication factor C subunit 1 protein is an important component of the DNA replication machinery and DNA damage response mechanism. Here we show the translocation of PfRFC1 from an intranuclear localization to the nuclear periphery indicating an orchestrated progression of distinct patterns of replication in the developing parasites. PfRFC1 responds to genotoxic stress via elevated protein levels in soluble and chromatin bound fractions.Reduction of PfRFC1 protein levels upon treatment with antimalarials suggests an interplay of replication and DNA repair pathways leading to cell death. Additionally, mislocalization of the endogenously tagged protein confirmed its essential role in parasites’ replication and DNA repair. This study provides key insights into DNA replication, DNA damage response and cell death in plasmodium falciparum.ImportanceFrontline drugs have been found to induce DNA damage in the human malaria parasite Plasmodium falciparum. The genotoxic stress response in Plasmodium and the interplay between DNA damage repair, replication and activation of programmed cell death pathways remains largely undescribed. This study shows a distinct pattern of localization of PfRFC1 during replication and DNA repair. PfRFC1 responds to genotoxic stress with an increase in protein expression. Interfering with the RFC complex formation or mislocalization of PfRFC1 is associated with disrupted genotoxic stress response. Additionally, a reduction of PfRFC1 protein levels is observed upon treatment with antimalarial drugs or under apoptosis like conditions, highlighting the role of DEVD/G like motif in mediating programmed cell death in these parasites. This study sheds light on the role of PfRFC1 in differentially responding to replication, genotoxic stress and programmed cell death in Plasmodium parasites.</jats:sec